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[THE BOTRYTIS BLIGHT OF TULIPS 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 



EDWIN F. HOPKINS 



TiBeprlnted from Memoir 45— Cornell University Agricultural Experiment Station 

August, 1921 



THE BOTRYTIS BLIGHT OF TULIPS 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 

EDWIN F. HOPKINS 



Reprinted from Memoir 45 -Cornell University Agricultural Experiment Station 

Aujiust, 1921 



U w -^civa^a;^ 









CONTENTS 

PAGE 

The host plants 315 

Varietal susceptibility 3I6 

The disease 3I6 

Names 316 

History and distribution ' 317 

Economic importance 318 

Symptoms 319 

On the bulbs 319 

On the leaves 323 

On the flowers 326 

On the stalks 328 

Etiology 328 

Nomenclature 328 

Morphology 332 

Physiology 335 

Growth 335 

Conidia production 338 

Dissemination of conidia 339 

Pathogenicity 339 

Experimental methods 340 

Results of inoculations 342 

Discussion of parasitism 349 

Life history 35O 

Primary inoculation and infection 350 

Secondary inoculations and infections 350 

Pathological histology 351 

Control 355 

Summary 357 

Bibliography 359 



311 



THE BOTRYTIS BLIGHT OF TULIPS 



Memoir 45 



Plate XXXIII 




TULIP PLANT AFFECTED WITH BOTRYTIS BLIGHT 
One-half natural size 



THE BOTRYTIS BLIGHT OF TULIPS ^ 

Edwin F. Hopkins 
THE HOST PLANTS 

It is reasonably certain that the Botrytis blight is restricted to plants 
in the genus Tulipa. This genus includes Tulipa Gesneriana L., the 
Darwin, or late, tulips, T. suaveolens Roth., the Due van Thol tulip, 
embracing the early and forced varieties, and T. sylvestris L., the so- 
called wild tulip. The members of this genus have been under cultivation 
for so long that it is difficult to refer them to any natural species although 
the arrangement just given is commonly accepted (Bailey, 1917 : 3393-3394). ^ 

Numerous references to the occurrence of this disease on other hosts 
have appeared in the literature. However, most of these statements are 
based on insufficient evidence, and show that the author had under 
consideration another Botrytis disease and did not attempt to verify his 
conclusions by cross inoculations. Ritzema Bos (1903 a: 20), for instance, 
s.iys that while the disease affects other bulbs, such as hyacinths, gladioli, 
and certain iris species, the tulip is by far the most susceptible. Klebahn 
(1905:15-17) takes exception to this statement since he has found the 
disease on none of these plants except the tulip; furthermore, his experi- 
ments show the hyacinth to be immune. He seems to infer that Ritzema 
Bos was confusing two different diseases, and that possibly, on these 
hosts, the disease with which he was dealing was caused by Sclerotium 
TuUparum Klebahn. However, Klebahn states that practical gardeners 
have told him that on ground which had borne bulbous begonias and 
dicentra no tulips came up. 

Other notes frequently appear by authors who evidently confuse 
diseases caused by other species of Botrytis with the one under con- 
sideration, which is caused by Botrytis Tulipae (Lib.) comb. nov. Halsted 
(1891:352) gives a good example of this in attributing the disease on 
onions to this organism. 

1 Also presented to the Faculty of the Graduate School of Cornell University, March, 1920, as a major 
thesis in partial fulfillment of the requirements for the degree of doctor of philosophy. 

Acknowledgment. The author wishes to express his thanks to Professor H. H. Whetzel, of Cornell 
University, for his many valuable suggestions and for his helpful advice during the progress of the work. 

2 Dates in parenthesis refer to Bibliography, pages 359 to 361. 

315 



316 . a^^^V Edwin F. Hopkins 

In order to gain some knowledge of the host range of the parasite 
concerned, numerous cross-inoculations were made by. the writer. These ■ 
experiments, which are summarized under the heading Pathogenicity^ 
(page 339), prove that the organism concerned does not attack certain 
of these hosts under artificial conditions, and therefore it probably would 
not under natural ones. Klebahn (1904, 1905, 1907) had previously 
made certain cross-inoculations tending to disprove the existence of a 
generalized type of parasitism in this pathogene. 

The disease is restricted, under normal conditions, to the genus Tulipa; 
and while similar Botrytis diseases occur on the other hosts mentioned, 
and even on the tulip itself, they are not identical with this one. 

VARIETAL SUSCEPTIBILITY 

Varieties of both the early tulips, Tulipa suaveolens, and the late tulips, 
T.*Gesneriana, are susceptible. Likewise, as already mentioned, the wild 
tulip, T. sylvestris, has been proved susceptible. The writer has collected 
diseased specimens of many varieties of the first two species and has seen 
the disease on the wild species. 

Klebahn (1905:11), in his experiments, tested five varieties of tulips, 
presumably early varieties, but finding all susceptible he drew no general 
conclusion from this result. He thinks it would be desirable to compare 
the susceptibility of the early and the late species. 

The writer found the disease on a large number of varieties of both 
species, and isolated the causal organism; he also succeeded in artificially 
infecting both species. He had almost concluded that there was not much 
difference in susceptibility. But in the spring of 1917, in a garden on 
the Cornell University campus where there was then a severe outbreak 
of the disease, one variety of late tulips (Baronne de la Tonnaye), which 
had certainly been exposed to the infection, showed no evidence of the 
disease. Up to the present time there has not been an opportunity to 
test this variety further. 

THE DISEASE 
NAMES 

Various names have been applied to the disease. Ritzema Bos (1903a: 
19) incorrectly used the name kwaden plekken, a term applied by Dutch 
bulb growers to soil that will not produce tulips. Later Klebahn (1907:3) 



The Botrytis Blight of Tulips 317 

showed that there are various causes of these "bad spots," but that 
usually they are due to Sderothim Tuliparum. Hence the term kwaden 
plekken may not be applied specifically to this disease. 

Ritzema Bos (1903 a: 19) uses also the word Umf alien to designate 
the disease as it occurs on tulip tops because it often causes the stem to 
break over. The writer believes, however, that this name is better applied 
to a physiological disease of tuHps described by Sorauer (1903:265). 
''Tulip mould" is the term used by Massee (1899:158) and also by 
Halsted (1902:438). Later Jacob (1912) states that it is commonly 
knovm as " fire." 

The writer would suggest the name Botrytis blight for this disease, 
since it causes a typical blight, and although there is another Botrytis 
disease of tulips, this is the more important one by far. 

HISTORY AND DISTRIBUTION 

The Botrytis blight of tulips was probably first described in 1830 by 
Madame Libert, in connection with an herbarium specimen (Crypt. Ard. 
No. 36). She evidently observed only the sclerotia. She describes the 
fungus from the sclerotia as Sclerotium Tulipae Libert. From her 
description it is evident that she had studied the sclerotia of Botrytis 
'parasitica Cavara (Saccardo, 1888-89). 

The disease was first carefully observed by Cavara (1888), in upper 
Italy. However, Ritzema Bos (1903 a: 26) thinks Wakker (1885:22) 
had the same disease under consideration before Cavara's publication 
appeared. He called it "tulpenziekte." Ritzema Bos (1903 a: 25) states 
that the disease had been known in Holland for more than twenty 
years, but that he is not certain when it first became seriously destruc- 
tive there. In 1890 affected tulip bulbs were sent to him from Norway. 
The work of Ritzema Bos, while in some respects not very accurate, 
was valuable in that it attracted attention to the importance of the 
disease. He began his studies in 1896 and published several papers 
on the subject. 

Carruthers (1901 : 246) notes the occurrence of the disease in Northamp- 
tonshire and Cambridge, England, in 1901. It was reported near St. 
Petersburg in 1911 (Elenkin, 1911). Klebahn (1904:18) mentions its 
appearance in Hamburg, Germany, in 1902, and in other parts of Germany 



318 Edwin F. Hopkins 

later. He has done some of the most important work in clearing up the 
confusion in regard to the various sclerotial diseases of tulips and their 
life histories. 

The appearance of the disease in America was first recorded by Halsted 
(1902). He had received in 1901, from a grower at Cape May, New Jersey, 
a diseased specimen, the bulb of which had originally been obtained 
from Holland. He stated that the disease had also occurred the previous 
season (1902:438). Obviously, therefore, it was introduced directly 
from Holland. 

In a letter to Professor H. H. Whetzel, of Cornell University, Professor 
W. J. Morse, of the Maine Agricultural Experiment Station, mentions 
some unpublished records of the appearance of the disease at Barrington, 
Nova Scotia, in 1904, at Cobourg, Ontario, in 1906, and at Cartine, Maine, 
in 1910. There are also later notes. Professor Whetzel and the writer 
have received or collected specimens from Amsterdam, Sassenheim, and 
Aalsmeer, in Holland; from Germany; from Ithaca, McGraw, Garden 
City, Jamaica, and Brooklyn, in New York State; from Madison, Wis- 
consin; from Manistee, Michigan; from Washington, D. C; from Belling- 
ham, Washington; and from Carthage, Missouri. There is no doubt 
that the disease is widespread throughout the United States and Canada, 
and it probably will be found wherever tulips are grown. 

ECONOMIC IMPORTANCE 

There are apparently no exact figures available regarding the amount 
of damage from the Botrytis blight. Both Ritzema Bos and Klebahn 
speak of the great loss which this disease occasions to both field growers 
and florists. In fact, Ritzema Bos (1903:91) was engaged for a time by 
a growers' association to investigate the disease. 

Elenkin (1911) reports that in 1911, near St. Petersburg, Russia, 50 
per cent of the tulip crop was destroyed by this and other tulip diseases. 
A collection and observations made by Whetzel at the New York Botanical 
Garden in 1916 show the Botrytis blight to have been severe there at that 
time. Occurrence of the disease is recorded also by Stout (1918:241), 
and letters and specimens from Dr. David Griffiths indicate that it was 
very prevalent in the Federal Government's bulb gardens at Bellingham, 
Washington, in 1917. 



The Botrytis Blight of Tulips 319 

In the spring of 1917 the writer observed the disease in an cpiphytotic 
condition. Most of the tuHp tops in the ornamental beds in the Cornell 
University campus were severely attacked. These were late, or Darwin, 
tulips, and counts made on one variety, Spathulata, showed 100 per cent 
of the leaves diseased, of which 33 per cent were strongly infected and 
67 per cent only slightly. Of the stalks, 98 per cent were diseased, 
23 per cent severely and 75 per cent slightly. The bulbs all showed 
slight infections, but it is uncertain whether these were infections by 
Botrytis Tulipae or by Penicillium sp. However, in the variety Spath- 
ulata 4.6 per cent of unmistakable Botrytis lesions were found, and 
in the variety Mrs. Grover Cleveland, 5.2 per cent. The presence of 
sclerotia in these lesions made identification certain. These were prob- 
ably lesions from the previous year and they show how small an 
amount of original inoculum is necessary to produce a severe infec- 
tion on the tulip tops. Accordingly it is impossible to lay too much 
emphasis on the selection of clean bulbs. The writer believes that this 
is the most important disease of the tulip in this country. 

SYMPTOMS 

On the bulbs 

On the brown outer skin, or husk, of the affected bulbs, small black 
sclerotia may frequently be found, about 1 millimeter in diameter. They 
appear also on the old, dried, flower stalk of the previous season, which 
sometimes remains attached to the bulb (fig. 22). The removal of this 
papery, brown skin often reveals lesions on the outer, white, bulb scale 
which might otherwise have escaped notice. These lesions vary from deep 
yellow to brown, are usually circular in outline, and have a definite margin 
which may be somewhat raised. The central part is ordinarily depressed 
and may have on its surface small black sclerotia (fig. 23). The lesions 
are formed sometimes at the apex of the bulb, sometimes at the base, 
but more often in the region between. Less frequently the sclerotia may 
appear white, which is due to their immaturity. By removing the outer, 
fleshy scale and examining its inner side, it will be seen that some of the 
lesions have penetrated almost to the inner surface. They rarely extend 
into the scales beneath. 



320 



" Edwin F. Hopkins 




1 



The Botrytis Blight of Tulips 



321 



Under warm, humid conditions, a large part of the outer scale may 
become affected, and in some cases it is so densely covered with sclerotia 
that these coalesce and form a crust. 

Care must be exercised not to confuse the lesions on the bulbs due to 
the very common Penicillium rot with those caused by Botrytis Tulipae. 




Fig. 23. botrytis blight lesions on the bulbs 

The lesions in the outer, fleshy, bulb scale are depressed and show sclerotia on their surface. 

Natural size 



This is especially true of the incipient lesions which, in these two diseases, 
are often found almost impossil^le to differentiate. In general, the lesions of 
the Penicillium rot are more indefinite in outline and of a lighter yellow 
color, are usually raised and uneven, and do not, of course, have sclerotia 
on their surface. The appearance of green mold, which forms under favor- 
able conditions, is a distinguishing characteristic. 



322 



Edwin F. Hopkins 




Fig. 24. botrytis blight lesions on the leaves 
Young lesions. Three-fifths natural size 



The Botrytis Blight of Tulips 



323 



On the leaves 
On the leaves the lesions show first as minute, yellowish spots, some- 
what elongate in the direction of the leaf veins and surrounded by a darker. 




Fig. 25. botrytis blight lesions on the leaves 
Lesions of a more advanced stage of development. One-half natural size 

water-soaked area. They are slightly sunken and give the leaf a speckled 
appearance. As they enlarge, the areas become more depressed, the 



324 



Edwin F. Hopkins 





Fig. 26. botrytis blight lesions on the leaves 
Peculiar twisting of leaves due to a marginal lesion. Natural size 



The Botrytis Blight of Tulips 



325 



color changes to a whitish 
gray with a brownish tinge, 
and a translucent or water- 
soaked area appears about 
the margin. At this stage 
the margins of the lesions 
are quite definite (fig. 24). 
Toward the center abundant 
conidiophores are often pro- 
duced. Under favorable 
conditions the lesions enlarge 
still farther, coalesce, and 
frequently involve the entire 
leaf. If a lesion develops 
toward the base of a leaf, 
it may cause the leaf to 
break over. When an infec- 
tion takes place on the mar- 
gin of the leaf near the tip, 
there results the character- 
istic appearance shown in 
figure 26. This wrinkling 
and bending of Ihe leaf to 
one side is due to the more 
rapid growth of the healthy 
tissue opposite the lesion. 
(Klebahn, 1905:4). 

Both young and old lesions 
are found on the same leaf 
showing that infections take 
place continuously (fig. 25). 
The outer sheathing leaf is 
likely to be attacked before 
the others, probably being 
infected as it emerges from 
the bulb. It bends down- 
ward and usually is abun- 
dantly covered with conidio- 
phores (fig. 27). 





Fig. 27. a diseased plant 

Showing the outer sheathing leaf infected by con- 
tact with a lesion at the tip of the bulb. Note the 
abundance of conidiophores which give rise to 
inoculum for secondary infections. Natural size 



326 



Edwin F. Hopkins 



On the flowers 

The lesions on the flowers (Plate XXXIII) are very striking, especially 
on red varieties of tulips. They begin as minute spots, whitish to light 
brown, the color being bleached from the perianth. These spots are 




Fig, 28. lesions on the flower 
Natural size 

evenly distributed over the surface and usually show no Botrytis 
fructification (fig. 28). After the lesions enlarge, however, they turn a 
deeper brown and involve the entire segment of the perianth, which 
finally becomes dry and wrinkled. At this time they show abundant 
conidiophores. The whole flower may be affected and appear blighted. 



The Botrytis Blight of Tulips 327 




Fig. 29. bud blight showing the blanching op the infected tissue 

Natural size 



328 Edwin F. Hopkins 

Indeed, this blighting may take place when the flower is still in the bud 
and prevent it from opening. Such a typical bud blight is reproduced 
in figure 29. 

On the stalks 

While the lesions on the stalks are still small, they have much the same 
appearance as those on the leaves except that they are more elongate 
and more depressed. They are of a light brownish color in the center, 
and are surrounded by a water-soaked area. Older lesions near the base 
of the stalk appear as dark brown patches and often bear sclerotia on 
their surfaces, while those higher on the stalk, usually originating in the 
leaf axils, are grayish white and are covered with conidiophores. Both 
this blanching effect and the conidial layer are well illustrated in 
figure 30. 

The extension of the lesion through the stem causes the latter to weaken 
at the point of attack and break over. If the lesion is near the base, 
the whole plant topples over; if it is higher up, the flower droops. 

ETIOLOGY 

Nomenclature 

The tulip disease under discussion is caused by Botrytis Tulipae (Libert) 
comb. nov. A sclerotial form, referred to the form genus Sclerotium by 
Madame Libert, in 1830, belongs to this species. She called it Sclerotium 
Tulipae and was the first to describe it (Crypt. Ard., no. 36). As 
previously mentioned, she apparently did not observe the conidial form. 
Her original description, which the writer has not been able to see, is 
taken from Klebahn (1907:5) who quotes it as follows: 

Sclerotium Tulipae, N. Sparsum, adnatum, parvum, ovale, pallide fiiscum, laeve, demura 
nigrum, rugosum, intus album. Ad caules, pericarpia et semina Tulipae Gesnerianae. 
Autumno. 

In 1836 the species was again designated as Sclerotium Tulipae by 
Weinmann (1836:647). Sometime between 1841 and 1859 it was once 
more described in connection with an herbarium specimen, this time 
by Westendorp. He called it Sclerotium entogenum (Herb, crypt. Belg., 
no. 827). Finally Cavara (1888) described the fungus more completely, 



The Botrytis Blight of Tulips 



329 



«i^j 





Ph 



330 Edwin F. Hopkins 

including both the conidial and the sclerotial stages, which he assumed 
belonged to the same species. His description is as follows: 

Botrytis parasitica nov. sp. Hyphis cinereis sparsis, erectis, articulo basali inflate; gonidiis 
ovatis, magnis, breviter pedicellatis, in ramulis minutis, capitatis, umbellatim dispositis; 
hyalinis vel dilute cinereis, 16-20 x 10-13 M. 

Forma scleroziale. 

Sclerotium Tulipae Lib. Haemisphaericum, vel oblongum, nigrum, vix rugosum, super- 
ficiale vel immersum, intus albidum f-l mm. latum. 

Hab. Ad folia, caules, petala, et capsulas Tulipae Gesnerianae in Horto botanico ticinensi. 

Cavara (1888:432) justifies his description of this species as a new one 
on the basis of morphological differences and also because of its strong 
parasitic action. In his discussion of nomenclature, he says that Sclerotium 
Tulipae Terry, which infects tulips in the south of France, according to 
Saccardo (1888-89,) is probably a synonym of Sclerotium cepivorum var. 
Tulipae Desm., and perhaps is the same as Scl. cepae Desm. Cavara was 
not able to compare specimens of Scl. Tulipae Terry with Scl. Tulipae Lib. 
The writer, however, had an opportunity to examine a specimen of the 
former at the herbarium of the New York Botanical Garden. This specimen 
consists of three or four sclerotia with no adhering plant material. The 
sclerotia are large, however, and are not those of Botrytis Tulipae (Lib.) 
comb. nov. At the same place the author was able to see some of the 
collection of Cavara designated Botrytis parasitica Cav., and found it to 
be identical with his own collections. 

Massee (1899:383) describes the organism as Sclerotinia parasitica. 
This description was not based on a perfect stage and in Massee's key 
is placed under " Conodial form only known." Since there is no evidence 
that the species under consideration is a Sclerotinia, this name is not 
valid. Massee's description is as follows: 

Sclerolinia parasitica, Massee; Botrytis parasitica, Cavara. Conidiophores grey, scattered, 
erect, basal joint inflated; conidia obovate, large, shortly pedicellate, on short umbellately 
arranged branchlets, hyaline or tinged grey, 16-21 x 10-13 u; sclerotia formed in the paren- 
chyma of the host, globose-depressed, smooth, greyish, then black, 2-3 mm. diam., sometimes 
numerous, and forming black crusts. 

Botrytis on leaves, stem, and flowers of cultivated tuUps; sclerotia more especially on 
the bulbs. 

Distr. Holland, Britain. 

A consideration of these facts has led to the following designation of 
the species: 



The Botrytis Blight of Tulips 331 

Botrytis Tulipae (Libert) comb, no v. 

Sclerotium Tidipae Lib. Crypt. Ard., no. 36. 1830. 

Sclerotium Tulipae Weinn. Hym. Ross., p. 647. 1836. 

Sclerotium entogenum West. Herb, crypt. Belg., no. 827. 1841-1859. 

Botrytis parasitica Cav. Appunti die Pathologia Vegetale. 1st. Bot. R. Univ. Pavia. 

Atti 2:1:432. 1888. 
Sclerotinia parasitica Massee. A text book of plant diseases, p. 383. 1899. 

Botrytis Tulipae (Libert) comb, no v. may be briefly described as follows : 

Mycelium variable in diameter, often anastomosing, branches not 
constricted at the base; conidiophores arising directly from the mycelium, 
erect, brown in color, proliferating, twisting on their axils when dry, 
slightly swollen at the base; branches of conidiophore arising at an angle 
of about 60 degrees, dichotomous, not streptiform, apices swollen; conidia 
large, 12-24 x 10-20 fx, obovate, raddish brown in mass; microscopically 
gray to hyaline, smooth with a short stalk, often or commonly not remaining 
attached; sclerotia at first white, finally black, small, 1-2 millimeters in 
diameter, circular or somewhat elliptical in outline, flattened vertically 
and often convex. Microconidia globose, about 3 ^^ in diameter, occurring 
on special penicillate, obclavate conidiophores arising in white tufts from 
the substratum (fig. 40). Parasitic on Tulipa spp. 

Cavara (1888:432) says that Sclerotium entogenum West, develops on 
the stalks of asparagus and does not differ greatly from Scl. Tulipae 
Lib.; he says, moreover, that Westendorp thinks Sclerotium entogenum 
West, should be regarded as the type of the species. Sclerotium Tuliparum 
Klebahn is a species which must not be confused with Botrytis Tulipae. 
It is a large, sclerotial form with which no conidial stage has yet been 
connected. 

The species must, therefore, still be classified among the Hyphomycetes 
of the Fungi Imperfecti. It is placed there by Lindau (1900:435) in the 
subgroup Mucedinaceae-Hyalosporae-Botry tideae . 

Klebahn (1904:21), from his studies on B. Tulipae, seems certain of 
the connection of the sclerotia occurring on the tulip bulbs with the 
conidial form on the leaves. However, as he did not use pure-culture 
methods, it seemed desirable to clear up this point. Pure cultures were 
made by the writer from the sclerotia occurring on the bulb and from 
conidia on the leaves of the same tulip plant. These cultures were identical, 
and when inoculated into sterilized tulip leaves both produced normal 
conidia and sclerotia. Both also caused infection of healthy tulip plants. 



332 



Edwin F. Hopkins 



Morphology 

The mycelium shows no peculiar characteristics. It varies in diameter, 
depending on the conditions under which it lives. The branches are not 

constricted where they join 
the parent hypha. Anasto- 
mosing is frequent (fig. 31). 
The conidi- 
ophores, 
when ma- 
ture, are 
deep brown 
in color ex- 
cept near 
the base, 
where they 
are hyaline. 
They are 
indetermi- 
n a t e in 
length, for 
under fa- 
vorable 
conditions, after 
forming one head 
of conidia termi- 




FlG. 31. MYCELIUM OF BOTBYTIS TULIPAE. X 600 

Type of branching and anastomosing of the hyphae 
(Camera-lucida drawing) 




Fig. 32. conidio- 
phore of botbytis 
tulipae. x 67 

A conidiophore 
unmounted. The 
several clusters of 
nally, the main axis conidia due to pro- 
may proliferate ^^^'^^^^^' ^^^ t^^ 
and form another 



twisting of the stalk 
when dry, are both 
visible. (Camera- 
lucida drawing) 



Fig. 33. swollen bases of conidiophores 
of botbytis tulipae. x 600 

(Camera-lucida drawing) 



head (fig. 50). This 
may be repeated 
until finally there are several clus- 
ters of conidia on a single conidio- 
phore (fig. 32). It should be remem- 
bered that although some of these 
clusters appear to be lateral, they 
are really formed terminally. WTien 



The Botrytis Blight of Tulips 



333 



dry, the conidiophore is 
flattened and twisted on 
its axis, and is slightly- 
swollen at the base (fig. 33) . 
The branches of the 
conidiophore rise from the 
main stalk at an angle of 
about 60 degrees, and their 
ultimate ends, which bear 
the conidia, are somewhat 
swollen. The conidia are 
produced on these swollen 
ends by a pushing out of 
the protoplasm in a bud- 
like manner, but they 

shortly assume a definite shape. When mature, they remain attached 
by short sterigmata (figs. 34 and 35). Various stages of conidial formation 




Fig. 34. 



ATTACHMENT OF CONIDIA 
TULIPAE. X 600 



OF BOTRYTIS 



Camera-lucida outline of conidia shown in fig. 35. 
Proliferation of the conidiophore has taken place after 
the formation of conidia on the head. 




Fig. 35. attachment of conidia of botrytis tulipae. X 840 
Note the short sterigma. These conidia are mature. (Photomicrograph) 



334 



Edwin F. Hopkins 




are shown in figures 36 and 37. ^ Usually young conidiophores were chosen 
for study because of the firmer attachment of the conidia. The prepa- 
ration of the mounts required considerable patience because of the delicate 
attachment of the conidia, and oftentimes many mounts were prepared 
before one was obtained which showed clearly the details of structure. 
When young, the conidia are hyaline, but as they mature they assume, 
in mass, a brownish color. Microscopical examination shows that most 

^ of this color is in the spore 

(y wall. The conidia are obo- 

- — vCV) vate, and when shed, the 

short sterigmata may some- 
times be seen still attached 
to the spores. 

The conidia vary con- 
siderably in length and to a 
less extent in width, but 
this variation does not de- 
part greatly from a mean 
which is more or less con- 
stant. This is shown graphic- 
ally in figure 38, which 
represents the measure- 
ments of one hundred spores. 
One curve expresses the vari- 
ation of the spore length 
and the other that of the 
spore width. 

The spores measured were from single-spore cultures and developed on 
sterilized tulip leaves in petri dishes. Abundant conidial fructifications 
were formed which were practically identical in appearance with those 
occurring in nature. They were mounted in the mounting fluid previously 
described^ and were measured under the oil-immersion lens by 

' In studying the detail of the conidiophore, especially the attachment of the conidia, satisfactory mounts 
were obtained by first adding to the material on the slide a drop of 70-per-cent alcohol in order to "wet" 
the conidiophores rapidly. The material was then flooded immediately with a mounting fluid prepared 
by mixing equal parts of 2-per-cent potassium acetate in water and 40-por-cent glycerin in alcohol and 
then adding a trace of copper acetate. The excess mounting fluid was removed with filter paper and the 
mount covered with a cover glass. Such mounts keep very well, do not dry out, and may be kept per- 
manently when ringed with balsam or gold size. 




Fig. 36. development of conidia of botbytis 
tulipae. x 600 

Illustrating the bud-like manner of their formation. 
(Camera-lucida drawing) 



The Botrytis Blight of Tulips 



335 




^o^ 



means of camera-lucida drawings. One hundred spores were outlined 
and the outHnes measured with a miUimeter rule. The error in measuring 
was calculated to be less than 2 per cent. On the basis of these measure- 
ments the average limits of variation may be placed at 12-24 x 10-20 ^i. 

These figures do not show the dis- 
tribution of the spores within these 
limits. From figure 38, however, it 
is apparent that the greater number 
have a length of 16-17 /x and a 
width of 9-10 IX. These measurements 
were checked with those of spores 
from another culture derived from a 
different locality. Measurements made 
of the spores of a large number of 
Botrytis specimens seem to show that 
one hundred conidia suffice to establish 
the mode for a given species. 

The sclerotia as formed in a petri- 
dish culture, (fig. 39) are at first white, 
and later, a shiny black. They are cir- 
cular, elliptical, or somewhat irregular 
in outline, flattened vertically, and 
often slightly convex. They might be described by the term "loaf -shaped." 
Ordinarily the sclerotia are about one millimeter in diameter. Their size 
may be considerably affected by the amount of drying to which they are 
subjected. 

Physiology 
Growth 

Botrytis Tulipae grows very readily on the various kinds of media, 

both liquid and solid, on which it has been planted. In the writer's 

experimental work the commonest medium employed was potato-dextrose 

agar. On this the fungus makes a rapid, fluffy mycelial growth, which 

later becomes appressed to the surface of the agar and the sclerotia then 

begin to form. These are very numerous and are imbedded in a tough, 

mycelial membrane which covers the surface of the substratum. As 

mentioned under Conidia production, conidia are rarely formed in such 

cultures. Moreover, there is scarcely any color production in this medium. 



Fig. 37. development of conidia of 
botrytis tulipae. x 600 

Mature conidia. (Camera-lucida drawing) 



336 



Edwin F. Hopkins 























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The Botrytis Blight of Tulips 



337 



It is of little importance to describe at length the growth on other 
media : sucrose is utilized as a source of carbon apparently as successfully 
as glucose; on a glucose solution, without mineral nutrition, the growth 
is very poor; on plain agar and water the growth is sparse and the mycelium 
tends to spread; but on agar containing a mineral-nutrient solution, with 




Fig. 39. sclerotia of botrytis tulipae 
As formed in a petri-dish culture on potato agar. Natural size 

no source of carbon, an excellent growth takes place, even better, perhaps, 
than that on potato-dextrose agar. This indicates that the carbohydrates 
of the agar itself are being utilized. With oxalic acid as a carbon source, 
the growth is poor. 

Growth is best on an acid medium, and, although no experiments have 
been made to determine the range of acidity, good development has 



1 



338 Edwin F. Hopkins 



always been observed when the acidity was approximately +20 according 
to Fuller's scale. Perhaps the range is as wide as that determined by 
Munn (1917: 407-108) for the growth of Botrytis Allii. 

Conidia 'production 

Although conidia are produced abundantly in nature on tulip leaves, 
in agar culture under the ordinary laboratory conditions they rarely 
appear. The fungus tends rather to form sclerotia. However, early in 
1917 the writer observed for the first time that conidia had formed under 
these conditions when some conidiophores developed in five petri-dish 
cultures. It should be noted that these conidia were produced without 
any special treatment, and that, although they were produced once or 
twice afterward, this is rather unusual under the conditions obtaining 
in ordinary culture vessels. 

On^March 17, 1917, sterilized tulip leaves in large test tubes, 20 x 2.5 
centimeters in size were inoculated with cultures of three different strains 
in duplicate. In two of these strains a fluffy mycelium soon developed, 
which spread along the leaves. After al^out ten days conidiophores began 
to develop, usually toward the top of the culture, and by March 31 there 
was an abundant development of conidiophores close to the surface of 
the leaves just beyond the aerial mycelium as well as on the mycelium 
itself. The conidia produced were able to cause infection in healthy 
tulips. This experiment was repeated on April 27, 1917, and the cultures 
were observed daily in order to note the first appearance of the conidia. 
This occurred in five cultures on May 8. 

Some time later, Professor Whetzel suggested that a partial drying of 
the plate cultures, after a good growth of mycelium had started, might 
produce conidia. This experiment was accordingly performed, and the 
partial drying was accomplished by so placing the petri-dish cover as to 
expose part of the agar surface, thus permitting more rapid evaporation. 
The petri dishes were usually left overnight in a dust-free chamber and 
were tightly covered again the next morning. Conidia were in this way pro- 
duced successfully in a large number of instances, not only from B. TuUpae, 
but also from other Botrytis species which do not readily fruit in culture. 
On halves of sterilized tulip bulbs in petri dishes the fungus was also 
found to fruit abundantly. There seems to be a relation between conidia! 



The Botrytis Blight of Tulips 339 

production and the relative humidity over the culture, or, to state it more 
concisely, a relation between the rate of evaporation and conidial pro- 
duction. 

There is, perhaps, something inherent in the nature of the host tissue 
which makes it an especially good substratum for the production of 
conidia; but it is more likely that the fungus finds in such cultures more 
variation in the moisture relations. This would explain why, in the 
experiments with sterilized tulip leaves, the conidia were not produced 
uniformly throughout the culture but only on a limited area toward the 
top of the culture tube, where the moisture relations were presumably 
most favorable. 

Dissemination of conidia 

The peculiar twisting of the conidiophores, already mentioned, is 
thought to have some relation to the dissemination of the conidia. In 
the first place, the conidia fall away very readily. This becomes obvious 
when an attempt is made to mount conidiophores bearing conidia in a 
liquid medium or if a conidiophore is jarred slightly. Secondly, the 
conidiophores are very hygroscopic, and a small change in the atmospheric 
moisture will cause them to twist with considerable violence and frequently 
even to dislodge the conidia. This phenomenon can be easily observed 
in this and in other Botrytis species by placing the specimen under a 
binocular microscope. Merely breathing on it gently suffices to produce 
these contortions. It may be concluded that in nature, owing to the 
frequent changes in humidity that occur, conidia are commonly dislodged 
in this manner and carried off by air currents. 

The conidia are usually wind-borne. The writer has found that 
inoculations with conidia are best made by either blowing or dusting 
them on the host plants and subsequently spraying water on them with 
an atomizer. 

Pathogenicity 

The pathogenicity of Botrytis Tulipae was first demonstrated by Klebahn 
(1905:6), who inoculated tulip bulbs with sclerotia from a pure culture. 
However, he appears not to have done much exact inoculation work, 
and, with the exception of the experiment cited, did not use pure cultures 
in his infection tests. The writer does not consider that Ritzema Bos 



340 Edwin F. Hopkins 

(1903 a: 24) demonstrated the pathogenicity of the organism by his investi- 
gations, for he did not use pure-culture methods, but merely developed 
Botrytis conidia on leaves in moist chambers and then dusted the conidia 
on sliced bulbs, causing the bulbs thus treated to decay. 

Experimental methods 

The writer made isolations of the fungus from material obtained from 
many localities in Holland, Germany, England, Canada, and the United 
States. These isolations were made both from dry specimens (for it was 
found that the fungus would retain its vitality for a long time without 
moisture) and from recently infected plants. Moreover, they were made 
from sclerotia, mycelium, and conidia from various parts of the host — 
bulbs, stalks, leaves, buds, perianth, and stamens. 

Although Klebahn (1905:12) found it difficult to obtain pure cultures 
from the sclerotia, the writer has experienced little difficulty in isolating 
from sclerotia by the following method: The sclerotium is rubbed free 
from all adhering material with a clean piece of cheesecloth, and is dipped 
for instant in 95-per-cent alcohol to remove the surface film of air, that is, 
to wet the surface. It is then placed in a 1 : 1000 mercuric-chloride solution 
for about thirty seconds, after which it is quickly removed with sterilized 
forceps and placed in a drop of sterilized water in a sterilized petri dish. 
To thoroughly remove the mercuric chloride, the sclerotium is then rinsed 
in several successive drops of sterilized water in the same petri dish. 
Usually six washings are sufficient. The sclerotium, thus prepared, is 
cut into four pieces and planted on a poured plate of potato-dextrose 
agar. The whole operation should not take more than five minutes. The 
writer has used this method in isolating several hundred Botrytis specimens 
as well as specimens of other fungi, and rarely has a contamination occurred. 

Fungi may be isolated from leaf tissue in this way if care is taken not 
to leave the material too long in the alcohol. In these experiments the 
mycelium was usually isolated from the leaf tissue and the stems by 
cleaning the epidermis with alcohol and then peeling it back, or often, 
when using stems and bulbs, by breaking or splitting them open so as to 
expose an uncontaminated surface. Small parts of the diseased tissue 
were then picked out with a sterilized, sharp-pointed scalpel and planted 
in agar. 



The Botrytis Blight of Tulips 341 

Conidia were isolated in several ways. Sometimes they were blown 
over the surface of the agar from the bent end of a platinum needle. The 
needle was attached to a piece of glass tubing which served as a blowpipe. 
The tendency at first was to gather too many spores on the needle, but 
with practice a sparse sowing was readily made and transfers were then 
obtained from the resulting colonies. Another method was to pick off, 
with sharp-pointed forceps, a single conidiophore, under a binocular 
microscope if possible, and then touch it to an agar plate in several places. 
Pure cultures usually resulted from some of these plantings, and often 
all the cultures were pure. 

Pure line cultures were obtained in two ways: first, by planting the 
fungus on a poured plate of plain agar and water, which caused the 
mycelium to spread out in its growth so that a single mycelial tip could 
be marked under the low power, cut off, and transferred; secondly, by 
the isolation of a single spore. In the latter method, which was the one 
most frequently employed, care was necessary lest more than a single 
spore should be obtained. A thin layer of agar containing a few conidia 
was poured into a petri dish and the spores were allowed to germinate 
slightly. After a conidium was marked and transferred to a poured 
plate, a microscopical examination was always made to ascertain positively 
that not more than one spore had been cut out. The growth of these 
cultures on potato agar is characteristic and is described under Physiology 
(page 355). 

Both mycelium and conidia were used as inoculum. The mycelium 
inoculum was prepared by growing the fungus in a petri-dish culture 
until the colony had reached the size of an inch or so in diameter. Small 
cubes of agar containing mycelium were then cut with a sterile scalpel 
from the edge of the colony and placed on the plant part to be inoculated, 
with the side containing the mycelium against the host. To prevent 
the inoculum from drying out, the plants were either placed in a large, 
moist chamber or covered with a bell glass or a lamp chimney. When 
it was desired to injure the inoculated parts, this was done by pricking 
a sterile, sharp-pointed scalpel through the agar block into the host tissue. 

In using conidia, difficulty was at first experienced in attempting to 
spray the plants with spore suspensions in water. No infections resulted. 
As already mentioned, this is explained by the fact that the conidia are 



342 



Edwin F, Hopkins 



not readily wet with water and consequently the water sprayed contained 
but few conidia in suspension. This was demonstrated by a microscopical 
examination of the drops on a slide sprayed with the suspension. Very 
few spores were found, and hence the chances for infection were slight. 
Since this method was unsatisfactory, an attempt was made to secure 
infection by dusting the conidia on with a camel's-hair brush. This 
proved to be very successful, and abundant infection resulted, both on 
dormant bulbs and on growing tulip plants. Plants inoculated in this 
manner were sprayed with sterile water from an atomizer and kept 
moist overnight. 



Results of inoculations 

The results obtained from inoculating dormant tulip bulbs with 
mycelium are shown in table 1. From this table it is clear that the 
dormant bulbs must be injured at the point of inoculation in order that 

TABLE 1. Results from Inoculating Dormant Tulip Bulbs with Mycelium 



Culture 


Number of bulbs 
inoculated 


Number of bulbs 
infected 




Injured 


Uninjured 


Injured 


Uninjured 


B. 298 


4 

2 
2 

2 

2 
2 

2 


2 


4 
2 
2 
2 
2 
2 
2 





B. 163 





B. 112 





B. 149 





B. XVII 





B. XXVII 





B. XXXII 









Total 


16 


8 


16 










* The arable numerals represent cultures from domestic sources, and the roman numerals, those of 
foreign origin. 

mycelium may infect them. Briefly stated, the entire sixteen of the 
dormant bulbs which had been injured were infected by the inoculation, 
whereas none of the eight uninjured bulbs were thus affected. On the 
other hand, when ten bulbs were inoculated just after the flowers had 
been cut, aU became infected, although only five were previously injured. 



The Botrytis Blight of Tulips 



343 



A summary of the results obtained from inoculating tulip stems with 
mycelium appears in table 2. From this table it is evident that the tulip 
stems inoculated with twelve different cultures of Botrytis Tulipae, five 



TABLE 2. Results from Inoculating Tulip Stems with Mycelium 


Culture* 


Number of stems 
inoculated 


Number of stems 
infected 




Injured 


Uninjured 


Injured 


Uninjured 


B. 298 

B. XXVII 


3 
4 
4 
4 
4 
4 
3 
3 
3 
3 
4 
1 


3 
5 
5 
5 
5 
5 
3 
3 
3 
3 
5 
2 


3 
4 
2 
4 
4 
4 
3 
3 
3 
3 
4 
1 


3 
3 


B. XXV 


2 


B. XXVI... 


5 


B. XVII 


^ 


B. XXXII 


5 


B. 112 

B. 143 


2 
3 


B. 149 


3 


B. 150 

B. 163 

B. 414 


3 
5 

1 


Total 


40 


47 


38 


40 



* The arabie numerals represent cultures from domestic sources, and the roman numerals, those of 
foreign origin. 

TABLE 3. Results from Inoculating Tulip Leaves with Mycelium 



Culture* 


Number of leaves 
inoculated 


Number of leaves 
infected 




Injured 


Uninjured 


Injured 


Uninjured 


B. XXVII 

B. XXV - 

B. XXVI 


1 


2 
2 
2 
2 
2 
2 
2 






2 
2 


B. XVII 

B. XXXII 


2 
2 


B. 163 


2 


B. 414 


1 






Total 


7 


14 


7 


11 







*The arable numerals represent cultures from domestic sources, and the roman numerals, those of 
foreign origin. 



344 



Edwin F. Hopkins 




from foreign sources and seven from domestic, showed thirty-eight in- 
fections out of forty when pricked with a needle after inoculation, and 
forty out of forty-seven when not injured in this way. 

A summary of the results obtained from inoculating tulip leaves with 
mycelium is given in table 3. All seven leaves showed infections when 
inoculated and injured; of the uninjured leaves, eleven out of fourteen 
showed infections. 

Tulip flowers were inoculated with mycelium, with results as given in 
table 4. The tulip flowers, like the leaves, were all seven infected when an 
injury was made at the point of inoculation. Out of fourteen not so 
injured after inoculation, twelve showed infections. 

TABLE 4. Results from Inoculating Tulip Flowers with Mycelium 



Culture* 



B. XXVII. 

B. XXV... 

B. XXVI.. 

B. XVII. . 

B. XXXII. 

B. 163. . .. 

B. 414. .. . 



Total . 



Number of flowers 
inoculated 



Injured 



Uninjured 



14 



Number of flowers 
infected 



Injured 



Uninjured 



12 



* The arabic numerals represent cultures from domestic sources, and the roman numerals, those of 
foreign origin. 

It should not be deduced that the lower proportion of infections in 
the uninjured leaves, stems, and flowers was owing to the inability of 
the fungus to penetrate uninjured tissue. The writer attributes it rather 
to experimental error; for the inoculum was more likely to be lost or 
dried out before infection had opportunity to take place than when it 
was placed at once in such intimate contact with the host tissue as was 
the case when the latter was injured. 

On April 3, 1917, six dormant bulbs were dusted with conidia from 
a pure culture of strain B. XXVII, and by April 25 five of these were 



The Botrytis Blight of Tulips 



345 



strongly infected and some showed aerial mycelium arising from the lesions. 
On April 18, 1917, ten, clean, dormant bulbs were inoculated in the same 
manner with conidia from a culture of strain B. XXVII. In several 
days, nine of these showed numerous spots or streaks, varying in color 
from yellow to brown, where the conidia had been sown. 

On March 30, 1917, at 5 p. m., four tulip leaves were dusted with conidia 
from a pure culture and the following morning at nine o'clock small water- 
soaked spots had appeared, a microscopical examination of the epidermis 
of which showed that the conidia had germinated and had penetrated 
the cuticle. On April 2 these spots showed a rusty color and were sur- 
rounded by translucent, water-soaked areas. 

On April 1, 1917, at 4 p. m., three tulip plants in pots were inoculated 
with conidia of strain B. XXVII and placed under a large bell glass. 
On April 2, at 9 p. m., all had developed a considerable number of infections, 
as shown by the large number of small, yellow spots. These spots later 
become larger and of a somewhat reddish cast. About five days later 
the plants were severely diseased and showed a mycelial growth on the 
leaf surface, ibout seven days thereafter conidia were formed. On 
April 12 some sclerotia were noted in the leaf tissue. 

On April 13, 1917, eight tulip plants were inoculated with conidia of 
strain B. XXVII and placed in a large moist chamber. On April 16 
all showed numerous yellow-to-reddish spots on the leaves. A few similar 
spots appeared on the stems. By April 18, the small spots had coalesced 
and appeared as large, reddish lesions, some of which were covered with 
abundant conidiophores. 

The inoculations with conidia are summarized in table 5. The plants 
were not mechanically injured at the time of inoculation. 



TABLE 5. Results from Inoculating Uninjured Tulip Plants with Conidia 



Experiment 


Culture 


Plant part 


Number 
inoculated 


Number 
infected 


16 


B. XXVII 


Bulb (dormant) 

Bulb (dormant) 

Leaves (detached) . . . 

Tops 

Tops 


6 

10 

4 

3 

8 


5 


18 


B. XXVII . . . . 


9 


14 


B. XXVII 


4 


15 


B. XXVII 


3 


17 


B. XXVII 


8 









346 



Edwin F. Hopkins 



In using mycelium as inoculum on the leaves and the stems, the lesions, 
after a short time, become as typical as those of the same age produced m 
in nature. In the beginning, however, although they show the char- 
acteristic yellowing, they are not exactly like natural lesions, for they 
assume the shape of the agar block used in the inoculation. As the infection 
spreads, the region about the inoculum becomes water-soaked, then ^ , 
depressed, and finally dried out. At the last stage, the lesion assumes ■ 

a dull gray color and produces fluffy 
mycelium and sometimes also a co- 
nidial layer. The lesions tend to 
elongate in the direction of the stem 
and the leaf. If an inoculation is 
made on the edge of a young leaf 
near the tip, the peculiar twisting 
described on page 323, under the 
heading Symptoms, results. The 
whole plant top may be involved as a 
result of such an inoculation (fig. 42). 
On the dormant bulbs, also, the lesions 
are typical (fig. 41) with a dark brown, 
shiny surface. In using conidia as 
inoculum, the lesions are typical from 
the start and are essentially as de- 
scribed on pages 319 to 328. 

With but few exceptions, when in- 
fection was positive, the fungus in 
these experiments was re-isolated in 
pure culture by one of the isolation processes described under Methods, 
and checked identically with the original culture. Usually the tissue- 
planting method was the one employed. 

In order to determine the range of parasitism of this species, a con- 
siderable number of experiments were made on both closely related and 
distantly related plants to find out whether Botrytis Tulipae is able to 
infect them. 

The general results of these experiments appear in table 6. Certain of 
these, however, should be discussed more fully. 




Fig. 40. 



MICEOCONIDIA OF 
TULIPAE. X 600 



(Camera-Iucida drawing) 



The Botrytis Blight of Tulips 



347 



TABLE 6. Results of Inoculation on Various Hosts from Inoculating Plants 
Nearly and Distantly Related to the Tulip 



Plant 



Lily of the valley 
Onion (leaves) . . . 

Onion (bulbs) 

Onion (steins) . . . 

Lilium sp 

Narcissus (leaves) 
Narcissus (leaves) 
Narcissus (stems) 
Hyacinth (leaves) 
Crocus (tops) .... 
Crocus (bulbs) . . . 

Gladiolus 

Peony 

Potato 

Golden seal 



Inoculum 



Mycelium 
Mycelium 
Mycelium 
Mycelium 
Mycelium 
MyceUum 
Conidia. . 
Mycelium 
Conidia . . 
Mycelium 
Mycelium 
Mycelium 
Mycelium 
Mycelium 
Mycelium 



Number inoculated 



Injured 



14 

3 

16 

2 

6 

11 



3 



7 

5 

10 

19 

21 

6 



Uninjured 



Number infected 



Injured 



Uninjured 



From table 6, the relation of B. 
Tulipae to certain plants is evident : 
it is not able to attack at all the lily 
of the valley, the lily, the gladiolus, 
the potato, or the goldenseal, and, 
probably, not the peony. However, 
this relation will be made clearer by 
a discussion of certain observations 
made during the attempts to cause 
infection on these plants. 

Inoculation of onion. — Leaves and 
stalks of onion plants were inoculated 
with three different cultures, one of 
these a typical culture of B. Tulipae. 
In those plants that were injured at 
the time of inoculation, strong infec- 
tion took place on the leaves but 
none occurred on the stems. On the 
uninjured plants there was no infec- 
tion. To serve as checks, other plants 
were inoculated at the same time with 




Fig. 41. result of artificial inoculation 
Lesion on dormant bulb. The bulb 
was injured at the point of inoculation. 
Natural size 



348 



Edwin F. Hopkins 



mycelium from a culture of the onion Botrytis, B. Allii Munn, and with 
a large sclerotial form of Botrytis from tulips — not B. Tulipae. From 
both of these inoculations a strong infection resulted on the leaves and a 
slight infection on the stem, in the uninjured as well as the injured 

plants. A similar experi- 
ment with mycelium from 
the same cultures was tried 
on onion bulbs, and here 
B. Tulipae produced no in- 
fection, while the other two 
species produced a strong 
infection, but only on the 
injured bulbs. It is inter- 
esting to note here that on 
the uninjured bulbs inocu- 
lated with B. Tulipae, pecul- 
iar depressions appeared in 
the bulb scale that at first 
seemed to be slight infec- 
tions. However, microscop- 
ical examination showed 
that the mycelial threads 
had not penetrated. They 
were merely superficial. 
Nevertheless, beneath the 
mycelium some epidermal 
cells and other deeper-lying 
cells had been killed. Mi- 
croscopical sections of the 
injured bulbs showed my- 
celium in the punctures 

made by the scalpel. It had not, however, penetrated laterally into the 

tissue, although some of this tissue had been killed. 
Inoculation of narcissus. — The result obtained on the narcissus was 

similar to that on the onion: strong infections appeared on the leaves 

9,nd slight ones on the stems when the inoculation was performed on 




Fig. 42. result of artificial inoculation 
The whole top is involved. Natural size 



The Botrytis Blight of Tulips 349 

mechanically injured plants. Uninjured plants were not infected. When 
conidia were used to inoculate narcissus, there was no sign of resultant 
injury, and microscopical examination of the epidermis showed the conidia 
to be present but not germinated. 

Inoculation of hyacinth. — Detached hyacinth leaves were inoculated 
with conidia from pure culture. On the fifth day after inoculation, small, 
yellowish, depressed spots appeared. Microscopical examination of these 
spots showed an abundance of germinated conidia but no penetration of 
the epidermis by their germ tubes. Moreover, the tissue beneath the 
epidermis showed no mycelium. 

Inoculation of crocus. — Slight infections were caused on mechanically 
injured crocus leaves by mycelium of B. Tulipae, but no infection occurred 
on uninjured leaves. A large sclerotial Botrytis from tulip caused no 
infection in either case. Very slight infections were produced on the 
papery scales of crocus bulbs. On injured bulbs, all the five inoculated 
were infected; on uninjured bulbs, only one out of five was infected. 
Microscopical sections of these lesions showed mycelium ramifying through 
the tissue, and disintegration of the cells was observed. 

Discussion of parasitism 

It is evident from these pathogenicity experiments that B. Tulipae 
is practically restricted to tulips. Although under certain conditions it 
attacks some closely related plants, even such infection occurs, almost 
invariably, only when there is an injury made at the point of inoculation. 
Furthermore, a large number of injured plants failed to become infected. 
When we consider the ease with which the tulip may be infected, whether 
mycelium or conidia be used as inoculum and whether the host plant 
be injured or uninjured, these apparent exceptions only make more evident 
its restricted parasitism. Indeed the writer believes that in those instances 
in which B. Tulipae is reported on other hosts, if the fungus were really 
that species, the infection took place on an injured part of the host plant. 
On the other hand, the pathogene shows gradation in parasitism in its 
feeble attempts to invade plants other than its normal host. First, there 
are plants such as the crocus, on which are produced only slight infections 
which do not spread. Next, there are plants such as the narcissus and 
the onion, on which the conidia do not even germinate and infection 



350 Edwin F. Hopkins 

by mycelium can begin only at an injured place. Again, there are plants 
such as the hyacinth, on which the conidia will germinate and cause 
local injury, without actually invading the plant. Finally, there is the 
tulip, in the case of which infections take place easily on uninjured plants. 
While this series is too incomplete to be conclusive, the tendency shown 
is clear, and further experiments in this direction would probably furnish 
additional evidence of the very limited range of the parasitism of B. 
Tulipae. 

Life history 

Primary inoculation and infection 

The fungus survives the dormant period of the bulb as mycelium or 
sclerotia and is planted with it in the fall. When the bulb starts into 
activity in the spring, the fungus starts also and sometimes spreads 
throughout the entire outer scale of the bulb. If the original infection 
is Hear the apex of the bulb, the shoot also is involved in the lesion and 
the mycelium growing from the bulb tissue infects the leaf tissue. This 
condition was frequently encountered in studying the disease and is 
well illustrated in figure 27. Usually it is only the outer, sheathing leaf 
that is diseased, although sometimes the whole shoot may be affected 
and fail to emerge from the soil. After growing in the leaf for a time, 
the mycelium emerges from the dead tissue and, if favorable conditions 
prevail, conidiophores and conidia are produced. These are formed 
on the aerial mycelium and also arise directly from the mycelium in the 
leaf. The unspecialized hyphae and the conidiophores which arise from 
the leaf emerge through the stomata, and in the specimens observed, only 
one came from each stoma. 

Secondary inoculations and infections 

The conidia, produced in great abundance on these first-infected leaves, 
furnish abundant inoculum for secondary inoculations. Although it is 
not improbable that they are also transported by such other agencies 
as insects, spattering rain, animals, and man, the conidia are for the 
most part scattered to the infection courts by means of the wind. 

The infection courts may be any part of the tulip plant except the roots. 
Conidia falling on these parts germinate very quickly under proper con- 
ditions. Experiments with conidia in tulip juice and in distilled water 



I 



The Botrytis Blight of Tulips 351 

produced successful germination. In the former there was a good develop- 
ment of germ tubes overnight, while in the latter germination took place 
but the development of the germ tubes was poor. This experiment 
was conducted at room temperature. The germ tubes penetrate directly, 
as discussed under the heading Pathological histology (page 351) and 
cause infection. Visible evidence of infection often appears within the 
short period of twenty-four hours, as was demonstrated in the patho- 
genicity experiments. Under conditions unfavorable for germination the 
conidia are able to retain their viability for some time, as the following 
experiment illustrates. 

Tulip material abundantly covered with conidia was collected on June 
12, 1917, at Ithaca, New York. It was kept under laboratory conditions 
and the capacity of the conidia for germination was tested on June 12, 
June 25, July 12, and August 2. Germination of conidia was obtained at 
all of these dates except the last. This shows that in a dry condition 
the conidia retain for several weeks their ability to germinate. The 
lesions caused by their infections soon enlarge and produce more conidio- 
phores and conidia, which in turn are capable of producing more infections. 
That these infections are continually taking place is evident from the 
presence of lesions of various ages on the same leaf (fig. 25). 

Conidia may be carried from badly diseased tops to the bulbs, perhaps 
being washed down by rain. Several specimens were collected which 
clearly showed this. Incipient lesions were found on both the stalks 
and the bulbs of such plants, showing how the inoculum works down to 
the bulbs. These lesions increase in size and sclerotia are produced. 
When the bulb becomes dormant the development of the lesion is arrested 
and the fungus is again ready for hibernation. There is no doubt that 
the sclerotia retain their vitality for a long period. In fact, isolations 
have been made from sclerotia which have been in a resting state for 
several years. 

Pathological histology 

The material was fixed in Flemming's, in chromo-acetic, and in Gilson's 
fluids, was embedded in paraffin, sectioned, and stained with both Heiden- 
hain's iron alum-haematoxjdon and Flemming's triple stain. Some 
difficulty was experienced in sectioning lesions on the bulbs because 
of the numerous, large, starch grains present. In order to study the 



352 



Edwin F. Hopkins 






'.4 



i^ 













-^ 






Fig. 43. lesion on the outer bulb scale 

Photomicrograph of cross section through lesion. The accumulation of starch in the cells, 
and an incipient sclerotium, are visible 

penetration, certain areas on the tulip leaves were marked with india 
ink, inoculated by dusting with conidia, and, after various intervals, 
cut out, killed, fixed, and stained. 

On the bulbs, typical necrotic lesions appear, which show a peculiar 
accumulation of starch about the diseased area. This is pictured in its 
general features in figure 43, and in more detail in figures 44 and 45, one 
of which shows a diseased area and the other a healthy one. These 
starch grains are heart-shaped and large. They react to iodine in potassium 
iodide in the usual way, and with Flemming's triple stain are colored a 
beautiful pink. Why they should accumulate in this manner about the 
lesion is not known. 



The Botrytis Blight of Tulips 



353 



The mycelium in the Ijulbous 
tissue is usually of small diame- 
ter and is both inter- and in- 
tracellular. Usually in that 
part of the tissue where the 
mycelium is advancing and 
the cells are not yet killed, it 
is intercellular, while in the 
older part of the lesion the 
hyphae penetrate into the cells 
as well as between them. The 
protoplasm of the cells at this 
stage is practically gone. The 
collapse of these empty cells 
causes the lesion to be de- 
pressed. Sclerotia sometimes 
form on the surface of the 
lesion. In figure 43 an incip- 
ient sclerotium may be seen. 





The amount of starch shown here may be 
compared with that shown in figure 44 



Fig. 44. starch accumulation in diseased 
TISSUE. X 277 

Starch cells in a diseased area of an outer bulb 
scale, showing numerous starch grains and 
intercellular mycelium. (Camera-lucida drawing) 



Vascular bundles in the bulb 
scale were markedly affected, and 
in one case the xylem had entirely 
disappeared while the phloem, 
though attacked, still remained 
in part. In another specimen the 
bundle had been disintegrated on 
the side toward the lesion. This 
involved the phloem, the cells of 
which stained a deeper blue. 
There was starch accumulation in 
this region. 

Penetration of the fungus into 
the leaf tissue has been observed. 
No appressoria are formed by the 
germ tubes, which instead pene- 
trate directly through the leaf 
surface, either through stomata or 



354 



Edwin F. Hopkins 




Fig. 4^5. penetration of leaf 
TISSUE. X 277 

Transverse section of epidermis. 
(Camera-lucida drawing) 



between epidermal cells (figs. 46, 47). The germ 
tubes have not been observed to penetrate 
directly through epidermal cells. It has been 
noted that penetration more often occurs where 
the conidia are more numerous. Probably this 
is because of the greater enzymatic action, 
which hydrolyzes the cuticular substance. 

The mycelium in the leaf, like that in the 
bulb, is both inter- and intracellular. This is 
shown in figures 48 and 49. After the fungus 
has developed for a time in the leaf tissue, a 
collapse of the cells results and causes the leaf 
to become much thinner in the diseased area. 
Here also, where the mycelium is still intercel- 
lular, the cells are not killed. There is injury 
caused in advance of the mycelium. This 
indicates the excretion of toxic or enzymatic 
substances by the pathogene. 
The writer thinks that the injury caused in this disease is not due to 
oxalic acid. Some experiments were made to determine what the nature of 
the injury from oxalic acid would 
be. Several plants were injected 
hypodermically with solutions of 
oxalic acid of various concentra- 
tions, and lesions were produced 
which strikingly resembled those 
caused by a fungus. Further- 
more, microscopical examination 
indicated that no fungus had 
been accidentally introduced. 
However, the concentrations 
were necessarily higher than 
those produced by fungi in cul- 
ture. The work of Brown (1915) 
seems to show that neither oxa- 
lates nor oxalic acid take part Fig. 47. penetration of leaf tissue, x 277 
in the toxicity of B. Cinerea, but Surface view. (Camera-lucida drawing) 




The Botrytis Blight of Tulips 



355 




Fig. 48. intercellular mycelium in leaf tissue. X 600 
The cells have not yet lost their protoplasm. (Camera-lucida drawing) 

that this toxicity is due to 
enzymatic action. 

CONTROL 

It has been impossible to 
carry control experiments far 
enough to justify making 
any definite recommenda- 
tions for the control of 
Botrijtis TuKpae. A con- 
sideration of the pathogene, 
however, makes it evident 
that elimination is probably 
of first importance. Clean 
bulbs, free from mycelium 
and sclerotia, should produce 
clean tulips, for it is most 
probable that in these forms 
the pathogene is carried on 
the bulbs. Although the dis- 
ease may possibly be attrib- 
uted to infested soil, it often 

occurs on tuhps grown in soil Fi«- 49. intracellular mycelium in leaf 

tissue. X 600 

m which heretofore no tulips m, n f *u i f a -a e * * 

^ The cells oi the leaf are devoid ot contents. 

have been grown. (Camera-lucida drawing) 




356 



Edwin F. Hopkins 



H 




Fig. 50. proliferation of conidiophore. X 840 
Showing new branches arising from conidial cluster. (Photomicrograph) 

Until further experiments have been made, soil treatment cannot be 
recommended. Carbolineum has been recommended in the literature as a 
disinfectant for soil (Elenkin, 1911). Klebahn (1904:33) criticizes this 
method unfavorably, saying that not even weeds will grow in soil thus 
treated. 

Experiments made at Madison, Wisconsin, in 1917, showed that spraying 
tuhps with bordeaux mixture 5-5-50 caused considerable injury to both 
the leaves and the flowers, besides giving them an unsightly appearance. 
Accordingly this treatment is not to be recommended. 

From the present knowledge of this disease the following measures 
seem advisable: 

1. Selection of clean bulbs, free from lesions and sclerotia. When 
the sclerotia occur only on the outer papery scale this should be removed 
and burned. It is well also to inspect the old stalk of the previous year, 
if this still remains attached, for it frequently bears sclerotia (fig. 22). 



The Botrytis Blight of Tulips 357 

2. Careful handling, to avoid injuring the bulbs, as infection takes 
place more readily on injured bulbs than on healthy ones. 

3. Storage of the bulbs under proper conditions of temperature and 
humidity. The temperature should be kept as low as possible without 
injury to the bulbs, preferably about 40° F. The humidity also should 
be low. These conditions are especially desirable, as they retard the 
development of any small lesions that may be present on the bulbs at 
the time of storage, and prevent the germination of any conidia that 
may be on their surface. 

4. Removal and destruction of diseased plants when they appear in 

the field or the beds. This will limit, if it does not entirely prevent, 

secondary infections. 

SUMMARY 

An investigation of the tulip disease caused by Botrytis Tulipae (Libert) 
comb. nov. shows that it is present throughout the United States and that 
it was probably introduced with the introduction of tulip bulbs. Reports 
of the disease show that it has been in this country at least since 1901. 

Under normal conditions this disease is restricted to the genus Tulipa 
and within this genus practically all varieties are susceptible. One instance 
of apparent immunity is the variety Baronne de la Tonnaye, which, during 
an epidemic of Botrytis blight, showed no evidence of the disease. 

Counts made in the spring of 1917, at Ithaca, on one variety of late 
tulips, Spathulata, showed 100 per cent of the leaves and 98 per cent 
of the stalks to be affected. Other varieties were similarly infected. 
These infections were traced to the bulbs, of which the variety Spathulata 
showed 4.6 per cent with unmistakable Botrytis lesions and the Mrs. 
Grover Cleveland variety, 5.2 per cent. 

The disease is easily recognized on the bulbs when the fungous sclerotia 
are present in the lesions. On the leaves, the flower stalks, and the flowers, 
a severe blighting frequently takes place. 

Studies of the literature and herbarium specimens show that the disease 
under consideration is to be ascribed to Botrytis Tulipae (Libert) comb, nov.* 

Cultural studies have demonstrated that both the small sclerotia on 
the bulbs and the conidial form on the leaves and other parts of the tulip 
plant are stages of one and the same fungus, namely, Botrytis Tulipae. 

«In recent literature the fungus has gone by the name Botrytis parasUica Cavara, but the specific name 
of Libert has pr.ority. 



358 Edwin F. Hopkins 

The morphology of the parasite has been investigated in some detail. 
The manner of formation and attachment of the conidia has been brought 
out, microconidia have been demonstrated for this species and conidial 
measurements show that while the variation in size is from 12-24 x 10-20, 
the greater number of spores measure 16-17 x 9-10. 

Conidial production, which rarely takes place in pure cultures under 
ordinary conditions, was found to occur abundantly when plate cultures 
were partially dried. Abundant conidia were also formed on sterilized 
tulip leaves in large test tubes. 

The parasitism of B. Tulipae has been fully demonstrated by numerous 
infections brought about by the use of pure cultures of the organism. 
Inoculations of other plants, both nearly and distantly related, while 
showing the parasite to be restricted to tulips, show also that the parasite 
exhibits a weak and varying degree of ability to attack other plants. 

Hibernation is by means of sclerotia which live over the winter on 
the "bulbs. Infection spreads from these bulbs to the developing shoots, 
where abundant conidia are produced. These primary lesions serve as 
the source of inoculum for secondary infection. The conidia produced 
in this manner retain their vitality for several weeks. 

Sections through lesions on the bulb show an accumulation of starch 
about the diseased area. In the penetration of the tissue by conidial 
germ tubes, no appressoria are formed and the germ tubes penetrate 
directly through the epidermis or through the stomata. 

Although extensive control experiments have not been made, it is recom- 
mended that clean bulbs, careful handling of bulbs, proper storage, and 
systematic removal and destruction of diseased plants in the field will 
largely hold the disease in check. 

Memoir 39, The Genetic Relations of Ilant Colors in Maize, the sixth preceding number in this series of 
pub'ications, was mailed on July 19, 1921. 



The Botrytis Blight of Tulips 359 



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